As both initiators and executioners of apoptosis, caspases serve as important drug targets for many major diseases such as neurological diseases, cardiovascular diseases, cancer, autoimmune diseases and diabetes. To date, apoptosis research highly desires cell permeable and sensitive caspase substrates for detecting caspase activation in live cells and animals. By utilizing Biotium's patented technology, we created a novel class green fluorescent caspse-3 substrate that is capable of detecting caspase activity within individual live cell in real-time, and also effectively monitoring nucleus morphology of apoptotic cells. This bi-functional caspase substrate allows studying heterogeneity of cell population during apoptosis, early detection of apoptotic cells and monitoring cellular apoptotic process kinetically. The overall goal of this Phase I SBIR grant application is develop a far-red fluorescent bi-functional caspase-3 substrate that can be used for multiplex detection together with existing popular fluorescent biomolecules such as FITC, Alexa 488, Rhodamine, Texas-red conjugates for immunostaining. [unreadable] [unreadable] Apoptosis or programmed cell death is a distinct, intrinsic cell death program that occurs in various physiological and pathological situations. Disruptions in this process play important roles in many major diseases such as neurological diseases, cancer, autoimmune diseases, diabetes, and cardiovascular diseases. As both initiators and executioners of apoptosis, caspases serve as important drug targets for these diseases. Among them, caspase-3 has been extensively studied for their important roles in apoptosis and necrosis in many tissues. The development of multi- color cell permeable and sensitive caspase substrates described in this project will allows studying caspase-3 activation in live cells and correlation of caspase-3 activation with other cellular events. They also provide novel tools for directly screening caspase inhibitors or activators in live cells. These novel caspase substrates will greatly facilitate our understanding of apoptosis and its relation to various diseases and the development of future therapeutics for apoptosis related diseases. [unreadable] [unreadable] [unreadable]